Magnetically stabilized fluidized bed chromatography columns have been used to concentrate and separate various substances, e.g., chemicals and pharmaceuticals. See U.S. Pat. No. 4,780,113 (Koslow), issued Oct. 25, 1988, which is incorporated herein by reference. The Koslow patent is directed to an isomobility focusing process of one component from a multi-component feedstream using a magnetically stabilized fluidized bed. The feedstream is introduced into the magnetically stabilized bed of magnetizable solids for which the component to be separated has an affinity, the solids descending countercurrently to an ascending flow of a fluidizing medium, the feedstream and the fluidizing medium together comprising a fluid phase within the column, under conditions wherein the component is concentrated within at least one isomobility focusing zone within the column. The isomobility focusing zone is the region within the column where an equilibrium is maintained between the velocity of the component in the fluid phase and the velocity of the component on the solid phase. Conditions of temperature, pH or salt concentration are adjusted such that the desired component is purified and concentrated as it is introduced into the column. The desired component is removed from the column as a sidestream or by elution with an eluent.
By varying certain process parameters, it is possible to establish conditions such that the movement of a given component of the feedstream in the fluid phase is equal to the movement of that component on the solid phase. If the two phases are moving in opposite directions, the component will have no net velocity, i.e., its movement in one direction with one phase being equal and opposite to its movement with the other phase.
The isomobility focusing process is demonstrated in FIG. 2, attached hereto. FIG. 2 depicts a three component feedstream wherein component P has an affinity to magnetizable adsorbent solids having a velocity less than zero, (i.e., a descending flow). The component's affinity to the solids is carefully balanced against the upflowing velocity of the fluidizing medium which has a velocity greater than zero, (i.e., an ascending flow), such that the velocity of component P is approximately zero. The velocities of components A and B are less than zero and greater than zero, respectively. Concentration of component P results due to the different velocities of the individual components within the column. That is, due to their net velocities components A and B will move out of the isomobility zone, whereas component P having a net velocity of approximately zero will remain within the isomobility zone.
Experience has shown that isomobility focusing (IMF) in a magnetically stabilized fluidized bed chromatography column has the following disadvantages: (1) the process results in diffusion tailings of the selected component outside the isomobility focusing zone which necessitates the use a longer chromatography column and shorter operating periods; and (2) isomobility focusing is somewhat difficult to control, (i.e., IMF requires continual adjustment of the conditions to maintain an equilibrium between the velocity of the component in the ascending fluid phase and the velocity of the component on the descending solid phase).
The present inventor has developed a process which makes use of the enhanced capabilities of a magnetically stabilized fluidized bed chromatography column and also overcomes the aforementioned disadvantages pertaining to isomobility focusing. In this regard, the present inventor has developed a process which separates and concentrates a desired component of a feedstream in a magnetically stabilized fluidized bed chromatography column by means of positive focusing.
Positive focusing provides the following advantages over isomobility focusing: (1) elimination of diffusion tailings, i.e., it actively resists diffusion of the selected component from the positive focusing zone; (2) permits the use of a much shorter column than isomobility focusing; (3) extends the system's operational period; and (4) easier to control.
Multi-component separations by means of a continuous chromatography column using sidestream withdrawal is discussed in Dr. Mark Burns' thesis, entitled "Continuous Affinity Chromatography Using a Magnetically Stabilized Fluidized Bed", University Microfilms Inc., Ann Arbor, Mich. In accordance with the aforementioned thesis, a sidestream from the column is used to adjust the liquid flow in chromatography column and balance a component at the entrance of the sidestream. This balancing of components is accomplished by the following principle. In the lower section of the column, the liquid flow rate is greater than the solids flow rate and the desired component tends to travel up the column. Above the sidestream, the liquid flow rate is less than the solids flow rate and the protein will travel down the column with the solids phase. Thus, the net effect is that the desired component is focused at the withdrawal point of the sidestream.
The problem with a sidestream withdrawal system is that since the feedstream is injected adjacent to the sidestream withdrawal, the other components to be separated within the system must travel past the sidestream withdrawal port to be removed at the top or bottom of the column. Thus, as the solute moves up through the column, over 25 percent of the other components are removed via the sidestream withdrawal port. Such contamination cannot be avoided in this type of sidestream withdrawal system, since at least one component in the multi-component system would have to travel past or through two or more ports. Furthermore, a system controlled by adjusting the sidestream withdrawal rate is incapable of acting in a batch mode and results in the production of highly contaminated separated components.
The present inventor has discovered that the use of positive focusing in place of isomobility focusing in the separation and concentration of a component from a feedstream in a magnetically stabilized fluidized bed chromatography column greatly enhances the overall performance of the system. Furthermore, the present invention produces positive focusing by adjusting various operating conditions such that the desired component is separated and concentrated within a positive focusing zone, wherein above the zone the velocity of the component is less than zero and below the zone the velocity of the component is greater than zero.
The present invention also provides many additional advantages which shall become apparent as described below.